Tricyclic compounds useful for inhibition of G-protein function and for treatment of proliferative diseases

ABSTRACT

Novel compounds of Formula   &lt;IMAGE&gt;   are disclosed. Also disclosed is a method of inhibiting Ras function and therefore inhibiting the abnormal growth of cells. The method comprises administering a compound of the Formula 1.0 to a biological system. In particular, the method inhibits the abnormal growth of cells in a mammal such as a human being.

BACKGROUND

International Publication Number WO92/11034, published Jul. 9, 1992,discloses a method of increasing the sensitivity of a tumor to anantineoplastic agent, which tumor is resistant to the antineoplasticagent, by the concurrent administration of the antineoplastic agent anda potentiating agent of the formula: ##STR2## wherein the dotted linerepresents an optional double bond, X' is hydrogen or halo, and Y' ishydrogen, substituted carboxylate or substituted sulfonyl. For example,Y' can be, amongst others, --COOR' wherein R' is C1 to C6 alkyl orsubstituted alkyl, phenyl, substituted phenyl, C7 to C12 aralkyl orsubstituted aralkyl or -2, -3, or -4 piperidyl or N-substitutedpiperidyl. Y' can also be, amongst others. SO₂ R' wherein R' is C1 to C6alkyl, phenyl, substituted phenyl, C7 to C12 aralkyl or substitutedaralkyl. Examples of such potentiating agents include11-(4-piperidylidene)-5H-benzo 5,6!cyclohepta 1,2-b!pyridines such asLoratadine.

Oncogenes frequently encode protein components of signal transductionpathways which lead to stimulation of cell growth and mitogenesis.Oncogene expression in cultured cells leads to cellular transformation,characterized by the ability of cells to grow in soft agar and thegrowth of cells as dense foci lacking the contact inhibition exhibitedby non-transformed calls. Mutation and/or overexpression of certainoncogenes is frequently associated with human cancer.

To acquire transforming potential, the precursor of the Ras oncoproteinmust undergo farnesylation of the cysteine residue located in acarboxyl-terminal tetrapeptide. Inhibitors of the enzyme that catalyzesthis modification, farnesyl protein transferase, have therefore beensuggested as anticancer agents for tumors in which Ras contributes totransformation. Mutated, oncogenic forms of ras are frequently found inmany human cancers, most notably in more than 50% of colon andpancreatic carcinomas (Kohl et al., Science, Vol. 260, 1834 to 1837,1993).

In view of the current interest in inhibitors of famesyl proteintransferase, a welcome contribution to the art would be compounds usefulfor the inhibition of famesyl protein transferase. Such a contributionis provided by this invention.

SUMMARY OF THE INVENTION

Inhibition of farnesyl protein transferase by tricyclic compounds ofthis invention has not been reported previously. Thus, this inventionprovides a method for inhibiting farnesyl protein transferase usingtricyclic compounds of this invention which: (i) potently inhibitfarnesyl protein transferase, but not geranylgeranyl protein transferaseI, in vitro: (ii) block the phenotypic change induced by a form oftransforming Ras which is a famesyl acceptor but not by a form oftransforming Ras engineered to be a geranylgeranyl acceptor; (iii) blockintracellular processing of Ras which is a farnesyl acceptor but not ofRas engineered to be a geranylgeranyl acceptor; and (iv) block abnormalcell growth in culture induced by transforming Ras.

This invention provides a method for inhibiting the abnormal growth ofcells, including transformed cells, by administering an effective amountof a compound of this invention. Abnormal growth of cells refers to cellgrowth independent of normal regulatory mechanisms (e.g., loss ofcontact inhibition). This includes the abnormal growth of: (1) tumorcells (tumors) expressing an activated Ras oncogene; (2) tumor cells inwhich the Ras protein is activated as a result of oncogenic mutation inanother gene; and (3) benign and malignant cells of other proliferativediseases in which aberrant Ras activation occurs.

The compounds useful in the claimed methods are novel compoundsrepresented by Formula 1.0: ##STR3## or a pharmaceutically acceptablesalt or solvate thereof, wherein: (1) R¹ is a group selected from:##STR4## R² is selected from: (1) H, (2) C₁ to C₈ alkyl, (3) C₂ to C₈alkenyl, (4) C₂ to C₈ alkynyl, ##STR5## wherein said alkyl, alkenyl, oralkynyl is optionally substituted with one or more groups independentlyselected from:

(a) awl, aralkyl, heteroaryl, heteroarylalkyl or heterocycloalkyl; saidawl, aralkyl, heteroaryl, heteroarylalkyl or heterocycloalkyl optionallysubstituted with one or more groups independently selected from:

(1) C₁ to C₄ alkyl,

(2) (CH₂)_(t) OR⁸ wherein t is 1 to 4,

(3) (CH₂)_(t) NR⁸ R⁹ wherein t is 1 to 4, or

(4) halogen,

(b) C₃ to C₆ cycloalkyl,

(c) --OR⁸,

(d) --SR⁸,

(e) --S(O)R⁸,

(f) --SO₂ R⁸,

(g) --NR⁸ R⁹,

(h) ##STR6## R³ is selected from H, halogen or C₁ to C₆ alkyl (e.g.,methyl);

R⁴ is selected from H, halogen or C₁ to C₆ alkyl (e.g., methyl);

R⁵ is selected from: H, ##STR7## R⁶ is selected from H or C₁ to C₆ alkyl(preferably methyl or ethyl);

R⁷ is selected from H, C₁ to C₆ alkyl, haloalkyl, or --C(O)R¹¹ whereinR¹¹ is selected from C₁ to C₆ alkyl, C₁ to C₆ alkoxy or --NHR¹² (whereinR¹² is C₁ to C₆ alkyl or H), or R⁷ is an acyl radical of a naturallyoccurring amino acid;

R⁸, R⁹ and R¹⁰ am independently selected from H, C₁ to C₄ alkyl, C₃ toC₆ cycloalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, aryl oraralkyl; said alkyl, cycloalkyl, heteroaryl, heteroarylalkyl,heterocycloalkyl, aryl or aralkyl are optionally substituted with C₁ toC₄ alkoxy, aryl, heteroaryl, heterocycloalkyl, cyclopropyl, halogen,--OH, --C(O)R¹³, --SO₂ R¹³, or --NR¹⁴ R¹⁵ wherein R¹³ is selected fromC₁ to C₄ alkyl or aralkyl, and wherein R¹⁴ and R¹⁵ are independentlyselected from H, C₁ to C₄ alkyl or aralkyl; with the proviso that R⁸ isnot H in substituents (e), (f) or (k), and with the proviso that R⁹ isnot H in substituent (h) or (n), and with the proviso that R⁸, R⁹, orR¹⁰ is not --CH₂ OH or --CH₂ NR¹⁴ R¹⁵ when R⁸, R⁹, or R¹⁰ is directlyattached to a heteroatom (e.g., O, S or N).

optionally, when R⁸ and R⁹ are bound to the same nitrogen, R⁸ and R⁹,together with the nitrogen to which they are bound, form a 5 to 7membered heterocycloalkyl ring;

optionally, when R⁹ and R¹⁰ are bound to the same nitrogen, R⁹ and R¹⁰,together with the nitrogen to which they are bound, form a 5 to 7membered heterocycloalkyl ring;

--represents an optional bond;

W is selected from CH when the optional bond is present, or O, S or CH₂when the optional bond is absent;

X is selected from CH or N; and

Y is selected from N or CH.

This invention also provides a method for inhibiting tumor growth byadministering an effective amount of the tricyclic compounds, describedherein, to a mammal (e.g., a human) in need of such treatment. Inparticular, this invention provides a method for inhibiting the growthof tumors expressing an activated Ras oncogene by the administration ofan effective amount of the above described compounds. Examples of tumorswhich may be inhibited include, but are not limited to, lung cancer(e.g., lung adenocarcinoma), pancreatic cancers (e.g., pancreaticcarcinoma such as, for example, exocrine pancreatic carcinoma), coloncancers (e.g., colorectal carcinomas, such as, for example, colonadenocarcinoma and colon adenoma), myeloid leukemias (for example, acutemyelogenous leukemia (AML)), thyroid follicular cancer, myelodysplasticsyndrome (MDS), bladder carcinoma and epidermal carcinoma.

It is believed that this invention also provides a method for inhibitingproliferative diseases, both benign and malignant, wherein Ras proteinsare aberrantly activated as a result of oncogenic mutation in othergenes--i.e., the Ras gene itself is not activated by mutation to anoncogenic form--with said inhibition being accomplished by theadministration of an effective amount of the tricyclic compoundsdescribed herein, to a mammal (e.g., a human) in need of such treatment.For example, the benign proliferative disorder neurofibromatosis, ortumors in which Ras is activated due to mutation or overexpression oftyrosine kinase oncogenes (e.g., neu, src, abl, Ick, and fyn), may beinhibited by the tricyclic compounds described herein.

The compounds of this invention inhibit farnesyl protein transferase andthe farnesylation of the oncogene protein Ras. This invention furtherprovides a method of inhibiting ras famesyl protein transferase, inmammals, especially humans, by the administration of an effective amountof the tricyclic compounds described above. The administration of thecompounds of this invention to patients, to inhibit farnesyl proteintransferase, is useful in the treatment of the cancers described above.

The tricyclic compounds useful in the methods of this invention inhibitthe abnormal growth of cells. Without wishing to be bound by theory, itis believed that these compounds may function through the inhibition ofG-protein function, such as ras p21, by blocking G-proteinisoprenylation, thus making them useful in the treatment ofproliferative diseases such as tumor growth and cancer. Without wishingto be bound by theory, it is believed that these compounds inhibit rasfarnesyl protein transferase, and thus show antiproliferative activityagainst ras transformed cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms are used as defined below unlessotherwise indicated:

Ac--represents acetyl;

acyl radical of a naturally occurring amino acid--means a group of theformula --C(O)C(NH₂)R²⁶ R²⁸, i.e.: ##STR8## wherein R²⁶ and R²⁸represent the substituents of the amino acid bound to the α-carbon; forexample R²⁶ and R²⁸ can be independently selected from H, alkyl, oralkyl substituted with an R³⁰ group, wherein R³⁰ can be, for example,--OH, SH, --SCH₃, --NH₂, phenyl, p-hydroxyphenyl, indolyl or imidazolyl,such that HO--C(O)C(NH₂)R²⁶ R²⁸ is an amino acid selected from, forexample, alanine, cysteine, cystine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, serine, tryptophane,tyrosine or valine;

alkyl--(including the alkyl portions of alkoxy, alkylamino anddialkylamino)--represents straight and branched carbon chains andcontains from one to twenty carbon atoms, preferably one to six carbonatoms;

alkenyl--represents straight and branched carbon chains having at leastone carbon to carbon double bond and containing from 2 to 12 carbonatoms, preferably from 2 to 6 carbon atoms and most preferably from 3 to6 carbon atoms;

alkynyl--represents straight and branched carbon chains having at leastone carbon to carbon triple bond and containing from 2 to 12 carbonatoms, preferably from 2 to 6 carbon atoms;

aralkyl--represents an alkyl group, as defined above, wherein one ormore hydrogen atoms have been replaced by aryl groups, as defined below(e.g., benzyl);

aryl (including the aryl portion of aryloxy and aralkyl)--represents acarbocyclic group containing from 6 to 15 carbon atoms and having atleast one aromatic ring (e.g., aryl is a phenyl ring), with allavailable substitutable carbon atoms of the carbocyclic group beingintended as possible points of attachment, said carbocyclic group beingoptionally substituted (e.g., 1 to 3) with one or more of halo, alkyl,hydroxy, alkoxy, phenoxy, CF₃, amino, alkylamino, dialkylamino, --COOR¹⁶(wherein R¹⁶ represents H, alkyl, aryl or aralkyl (e.g., benzyl)), or--NO₂ ; and

Bu--represents butyl;

cycloalkyl--represents saturated carbocyclic rings branched orunbranched of from 3 to 20 carbon atoms, preferably 3 to 7 carbon atoms;

Et--represents ethyl;

halogen (halo)--represents fluoro, chloro, bromo and iodo;

haloalkyl--represents an alkyl group, as defined above, wherein one ormore hydrogen atoms have been replaced by halogen atoms;

heterocycloalkyl--represents a saturated, branched or unbranchedcarbocylic ring containing from 3 to 15 carbon atoms, preferably from 4to 6 carbon atoms, which carbocyclic ring is interrupted by 1 to 3hetero groups selected from --O--, --S-- or --N-- (suitableheterocycloalkyl groups include 2- or 3-tetrahydrofuranyl, 2- or 3-tetrahydrothienyl, 2-, 3- or 4-piperidinyl, 2- or 3-pyrrolidinyl, 2- or3-piperizinyl, 2- or 4-dioxanyl, etc.);

heteroaryl--represents cyclic groups, optionally substituted with R³ andR⁴, having at least one heteroatom selected from O, S or N, saidheteroatom interrupting a carbocyclic ring structure and having asufficient number of delocalized pi electrons to provide aromaticcharacter, with the aromatic heterocyclic groups preferably containingfrom 2 to 14 carbon atoms, e.g., triazolyl, 2-, 3- or 4-pyridyl orpyridyl N-oxide (optionally substituted with R³ and R⁴), wherein pyridylN-oxide can be represented as: ##STR9## heteroarylalkyl--represents analkyl group (as defined above) wherein one or more hydrogen atoms havebeen replaced by heteroaryl groups (as defined above); and

Ph--represents phenyl.

Representative compounds of the present invention include: ##STR10##

For the compounds of this invention, W is preferably CH or CH₂, with CH₂being most preferred; Y is preferably N; X is preferably N; R³ ispreferably halogen, with Br, Cl or I being most preferred, and Cl beingeven more preferred; and R⁴ is preferably halogen, with Br, Cl or Ibeing most preferred, and Br being even more preferred.

Representative compounds of this invention include those wherein R¹ isselected from: ##STR11##

Representative compounds of this invention also include those wherein R¹is selected from: ##STR12##

Representative compounds of this invention include compounds wherein R¹is selected from: ##STR13##

Representative compounds of this invention further include compoundswherein R¹ is selected from: ##STR14##

Representative compounds of this invention also include compoundswherein R¹ is selected from: ##STR15## and usually R¹ is represented byFormula (e) or (f) above wherein R⁵ is hydrogen.

Those skilled in the art will appreciate that R¹ substituents (e), (f),(g) and (h) can exist as the disulfide substituents (w), (x), (y) and(z), respectively.

Preferably, R² is selected from H, --C₄ H₉, --CH₂ C₆ H₅, --CH₂ CH₂ OCH₃,--CH₂ CH₂ SCH₃, --CH₂ CH₂ O-n-C₃ H₇, --CH₂ CH₂ CH₂ OCH₃, ##STR16##

Lines drawn into the ring systems indicate that the indicated bond maybe attached to any of the substitutable ring carbon atoms.

Certain compounds of the invention may exist in different isomeric(e.g., enantiomers and diastereoisomers) forms. The inventioncontemplates all such isomers both in pure form and in admixture,including racemic mixtures. Enol forms are also included.

Certain tricyclic compounds will be acidic in nature, e.g. thosecompounds which possess a carboxyl or phenolic hydroxyl group. Thesecompounds may form pharmaceutically acceptable salts. Examples of suchsalts may include sodium, potassium, calcium, aluminum, gold and silversalts. Also contemplated are salts formed with pharmaceuticallyacceptable amines such as ammonia, alkyl amines, hydroxyalkylamines,N-methylglucamine and the like.

Certain basic tricyclic compounds also form pharmaceutically acceptablesalts, e.g., acid addition salts. For example, the pyrido-nitrogen atomsmay form salts with strong acid, while compounds having basicsubstituents such as amino groups also form salts with weaker acids.Examples of suitable acids for salt formation are hydrochloric,sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic,fumaric, succinic, ascorbic, maleic, methanesulfonic and other mineraland carboxylic acids well known to those in the art. The salts areprepared by contacting the free base form with a sufficient amount ofthe desired acid to produce a salt in the conventional manner. The freebase forms may be regenerated by treating the salt with a suitabledilute aqueous base solution such as dilute aqueous NaOH, potassiumcarbonate, ammonia and sodium bicarbonate. The free base forms differfrom their respective salt forms somewhat in certain physicalproperties, such as solubility in polar solvents, but the acid and basesalts are otherwise equivalent to their respective free base forms forpurposes of the invention.

All such acid and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the invention and all acid and basesalts are considered equivalent to the free forms of the correspondingcompounds for purposes of the invention.

The following processes may be employed to produce compounds of theinvention. Various intermediates in the processes described below can beproduced by methods known in the art, see for example, U.S. Pat. No.3,409,621, U.S. Pat. No. 5,089,496, WO89/10369, WO92/20681, WO93/02081,and WO95/00497; the disclosures of each being incorporated herein byreference thereto.

Compounds of the invention can be produced from ketones of Formula 3.0:##STR17## as described below. Compounds of Formula 3.0 are known or canbe prepared by the procedures described in U.S. Pat. No. 5,089,496,WO89/10369, WO92/20681, and WO93/02081. For example, intramolecularcyclization of a nitrile of Formula 4.0: ##STR18## using a strong acid,such as CF₃ SO₃ H, at a temperature of about -15° to about 100° C., toform an imine intermediate which is hydrolyzed with water or aqueousacid to form the ketone of Formula 3.0

Alternatively, intramolecular Friedel-Crafts acylation of an acidchloride of Formula 5.0: ##STR19## may also provide the desired ketoneof Formula 3.0. The reaction may be carried out under the usualFriedel-Crafts conditions in an inert solvent and in the presence of aLewis acid such as aluminium chloride. Acid 5 chlorides of Formula 5.0can be obtained by the hydrolysis of a compound of Formula 4.0 to thecorresponding carboxylic acid. Typically this can be done by heatingwith an aqueous acid (e.g., aqueous HCl), followed by conversion of theacid to the acid chloride of Formula 5.0 under standard conditions wellknown to those skilled in the art (e.g., by treating with SOCl₂ oroxalyl chloride).

Ketones of Formula 3.2 (i.e., compounds of Formula 3.0 wherein W is CH)can be prepared by heating a compound of Formula 3.1 (i.e., a compoundof Formula 3.0 wherein W is CH₂) with SeO₂ in acetic acid. ##STR20##

The ketone of Formula 3.0 is converted to the compound of Formula##STR21## wherein L is Cl by a procedure analogous to that described inU.S. Pat. No. 3,409,621. For example, the ketone of Formula 3.0 isreduced to the corresponding alcohol using reagents such as sodiumborohydride, and then the hydroxy group is converted to Cl by usingreagents such as benzene and thionyl chloride. One skilled in the artcan convert the hydroxy group to other leaving groups (e.g., Br, I,mesyloxy or tosyloxy). The compound of Formula 6.0 (wherein L is Cl) isreacted, at a temperature of about 25° to about 100° C., with a cyanidesalt (e.g., CuCN AgCN or NaCN) in a suitable organic solvent, such aspyridine or benzene, to produce the nitrile of Formula 7.0. ##STR22##

The nitrile of Formula 7.0 can be hydrolyzed to an acid (Formula 8.0wherein R²⁰ is H), or an ester (Formula 8.0 wherein R²⁰ is --CH₃).Hydrolysis can be accomplished using an aqueous acid (e.g., HCl), or anacid (e.g., p-toluenesulfonic acid or H₂ SO₄) and an alcohol(e.g.,methanol or ethanol). The hydrolysis is carried out at atemperature of about 25° to about 80° C. ##STR23##

Alternatively, the compound of Formula 6.0 is reduced to the compound offormula 6.1 with a reducing agent, such as sodium borohydride, and asolvent, such as ethanol. The reduction is conducted at a temperature ofabout 25° C. The compound of Formula 6.0 can also be reduced to thecompound of Formula 6.1 with zinc and acetic acid using a temperature ofabout 25° to about 100° C. (usually about 80° C.). ##STR24##

The compound of Formula 6.1 can be convened directly to a carboxylicacid of Formula 8.0 by treatment with a base such as n-butyl lithiumfollowed by carbon dioxide.

The compound of Formula 8.0 is then reacted with a compound of Formula9.0 to produce the compound of Formula 10.0. When the compound ofFormula 8.0 is an acid (i.e., R²⁰ is H), the reaction is conducted witha coupling reagent (such as a carbodiimide, e.g.,dicyclohexylcarbodiimide) in a suitable solvent (such as DMF, i.e.,N,N-dimethylformamide) at room temperature. When the compound of Formula8.0 is an ester (i.e., R²⁰ is --CH₃), the reaction is conducted in thepresence of a base (e.g., triethylamine) in a suitable solvent (e.g.,DMF) using elevated temperatures (e.g., about 100° C.). ##STR25## BOC ist-butyloxycarbonyl.

Those skilled in the art will appreciate that the compound of Formula9.0 can exist as the two enantiomers ##STR26## and preferably theenantiomer of Formula 9.1 is used to make the compounds of theinvention. When the compound of Formula 9.1 is used compounds of formula1.1 are obtained.

The compound of Formula 10.0 can be deprotected (i.e., the BOC groupremoved) by treatment with an acid (e.g.,trifluoroacetic acid, orHCl-dioxane) to produce the compound of Formula 10.1: ##STR27##

The compound of Formula 10.1 can be converted to the compound of Formula1.1, wherein X is N, by acylation or reductive alkylation.

Alternatively, the compound of Formula 9.0 can be reacted withcarbonyldiimiazole at about 0° C. using methylene chloride to produce acompound of Formula 11.0: ##STR28## The compound of Formula 6.1 can betreated with butyl lithium, and then reacted with the compound ofFormula 11.0 to produce the compound of Formula 10.0. The compound ofFormula 10.0 can then be deprotected as described above to produce thecompound of Formula 10.1. ##STR29##

Scheme 1 describes the synthesis of 2-substituted piperazines wherein R²is H, alkyl, alkenyl, or alkynyl. Scheme 1 also describes the synthesisof 2-substituted piperazines wherein R² is alkyl, alkenyl, or alkynylwhich are substituted with substituent groups (a), (b), (c), (d) and (g)as defined above, with the exception that R⁸ and R⁹ can not be a groupthat is substituted with --C(O)R¹³ or --SO₂ R¹³. In Scheme 1,BOC-protected amino acids (12.0) are available commercially or can bemade by procedures well known in the art. These amino acids can becoupled (step 1) to a commercially available N-benzylglycine ethyl esterusing suitable coupling agents such as DCC (dicyclohexylcarbodiimide) orEDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) insuitable solvents (e.g., N,N-dimethylformamide, chloroform or methylenechloride) to produce a compound of Formula 13.0. Generally, thisreaction is conducted at room temperature (i.e., about 25° C.). The BOCprotecting group is removed (step 2) at room temperature with suitablereagents such as trifluoroacetic acid, or hydrogen chloride inchloroform or dioxane. The deprotected dipeptide is cyclized (step 3)under basic conditions to produce the compound of Formula 14.0. Thecompound of Formula 14.0 is then reduced (step 4) using LiAlH₄ inrefluxing ether (diethyl ether) or THF to give the piperazine of Formula15.0. The unsubstituted nitrogen of the piperazine of Formula 15.0 isprotected (step 5) with a BOC group by procedures well known in the artto give the compound of Formula 16.0. The N-benzyl group is removed(step 6) by catalytic hydrogenation (e.g., using Pd/C and hydrogen gasunder pressure of about 60 psi) to give the compound of Formula 9.0.##STR30##

Compounds of Formula 9.0, wherein R² represents alkyl, alkenyl oralkynyl substituted with (a), (c), (d) or (g) groups wherein R⁸ or R⁹are substituted with --C(O)R¹³ or --S(O)₂ R¹³ are made according to theprocess of Scheme 2. Compounds of Formula 9.0, wherein R² represents--C(O)NR⁸ R⁹ or --C(O)OR⁸, or wherein R² represents alkyl, alkenyl oralkynyl substituted with a group (e), (f), or (h)-(o) are also madeaccording to the process of Scheme 2. Compounds of Formula 17.0 (whereinR²² is an alkyl, alkenyl or alkynyl group containing either a --OHgroup, a --COOH or its corresponding ester) are available commerciallyor can be made by procedures known in the art. In Scheme 2, the compoundof Formula 17.0 is reacted according to the procedures described forScheme 1 (steps 1 to 4) to produce a compound of Formula 19.0 whereinR²³ is a hydroxy substituted alkyl, alkenyl or alkynyl group. Thecompound of Formula 19.0 is then protected with a BOC group and thendebenzylated according to the procedures in Scheme 1 (Steps 5 and 6) toproduce a compound of Formula 9.3. The unsubstituted nitrogen of thecompound of Formula 9.3 is protected (step 7) with a CBZ group(benzyloxycarbonyl) by procedures known in the art to produce thecompound of Formula 9.4.

When R²³ is --CH₂ OH, the hydroxy group can be oxidized to produce thecorresponding carboxyl group-(COOH). This carboxyl group can them beesterified to produce compounds wherein R² is --C(O)OR⁸, or the carboxylgroup can be converted to amides to produce compounds wherein R² is--C(O)NR⁸ R⁹ by procedures well known in the art.

To produce compounds of formula 9.0 in Scheme 2 wherein R² is asubstituent other than --C(O)OR⁸ or --C(O)NR⁸ R⁹ (i.e., substituents (5)and (6)), the hydroxy group on R²³ can be converted to a leaving group,such as chloro, mesyloxy or tosyloxy, by techniques well known in theart. Then the leaving group can be displaced by various nucleophilessuch as organometallics (to produce R² with an (a) substituent), thiols(to produce R² with a (d) substituent), sulfenyls (to produce R² with an(e) substituent), sulfinyls (to produce R² with an (f) or (m)substituent), amines (to produce R² with a (g) substituent), andalcohols (to produce R² with a (c) substituent). The hydroxy group onR²³ can also be acylated (to produce R² with a (j) or (k) substituent)or alkylated (to produce R² with a (c) substituent). When R²³ is alkylhaving more than one carbon atom, or alkenyl or alkynyl, the hydroxygroup can be oxidized, as discussed above, to produce the correspondingcarboxyl group (i.e., substituent (o) wherein R⁸ is H). This carboxylgroup can be esterified to produce compounds wherein substituent (o) is--C(O)OR⁸ wherein R⁸ is other than H, or converted to amides to produceto produce R² with an (l) substituent by procedures well known in theart. When the leaving group is displaced by an amine (e.g., --NR⁸ R⁹),the amine can then be converted to R² substituent groups (h), (i) or (n)by reacting the amine with an acyl halide (to produce R² with an (h)substituent), a carbamyl halide (to produce R² with an (i) substituent)or a sulfonyl halide (to produce R² with an (n) substituent) byprocedures well known in the art.

The preparation of compounds of Formula 9.0 is described in WO95/00497,published Jan. 5, 1995, the disclosure of which has already beenincorporated herein by reference thereto.

Compounds of Formula 1.0 wherein X is CH, and R² is alkyl, alkenyl oralkynyl, or R² is alkyl, alkenyl or alkynyl substituted withsubstituents (a), (b), (c), (d), or (g) with the exception thatsubstituents R⁸

or R⁹ cannot have a halogen, --OH, --C(O)R¹³ or --SO₂ R¹³ substituent,can be made from compounds of the Formula 22.0: ##STR31##

Compound 22.0 can be made according to the process: ##STR32##

The substituted piperidines of Formula 22.0 may be prepared, as racemicmixtures, by essentially the same methods as described in D. L. Cominsand J. D. Brown, Tetrahedron Letters, vol. 27 No. 38, pgs. 4549-4552,1986. Thus, 4-methoxypyridine (23.0) may be converted using a variety ofalkyl Grignard reagents (wherein R² is as illustrated below) andbenzylchloroformate to the desired unsaturated ketopiperidines (24.0).Removal of the benzylcarbamoyl group with concomitant reduction of thedouble bond by catalytic hydrogenation yields the substitutedketopiperidines (25.0). Alternatively, the benzylcarbamoyl group can beremoved with either base or acid followed by metal hydride reduction ofthe double bond to produce the compound of Formula 25.0. Alkylation ofthe compound of Formula 25.0 with a suitable alkyl iodide such as methyliodide in the presence of sodium hydride gives then-alkylketopiperidines (26.0). Reduction of the compound of Formula 26.0with sodium borohydride affords the hydroxypiperidine of Formula 27.0.The compound of Formula 27.0 is reacted with a suitable chlorinatingagent such as thionyl chloride to afford the 4-chloropiperidine ofFormula 28.0 which may in turn be converted by reaction with magnesiuminto the compound of Formula 22.0.

The compound of Formula 22.0 is reacted with the compound of Formula7.0, described above, in a suitable solvent such as diethyl ether orTHF. The reaction is conducted at room temperature (about 25° C.) toabout 50° C. This reaction is then followed by aqueous acid hydrolysisto yield ketones of the Formula: ##STR33##

The N-methyl group on the piperidine ring can be converted to acarboethoxy group (--COOC₂ H₅) by reaction with excess ethylchloroformate in dry toluene containing triethylamine at a temperatureof about 80° C. This procedure is similar to that described in U.S. Pat.Nos. 4,282,233 and 4,335,036. The carboethoxy group can be removed byeither acid or base hydrolysis to give the compound of Formula 30.0:##STR34##

The compounds of Formula 30.0 are prepared as diasteromeric mixtures.Preferably, the diasteriomers are separated into single isomers byclassical resolution methods or by chiral HPLC to yield: ##STR35##

The compound of Formula 30.0, preferably 30.1, can be converted to thecompound of Formula 1.0 (preferably 1.1), wherein X is CH, by acylationor inductive alkylation.

Acylation of the compounds of Formulas 10.1 and 30.0 can carried out byreacting the compound of Formula 10.0 or 30.0 with the correspondingcarboxylic acid of the desired R¹ group with a coupling agent, such as acarbodiimide such as dicyclohexylcarbodiimide(DCC) or DEC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). The acylation reactioncan be carried out in a suitable organic solvent such as DMF, THF ormethylene chloride at a temperature of about -10° to about 100° C.,preferably at about 0° to about 50° C., and most preferably about roomtemperature. When the coupling reagent is DCC or DEC, the reaction ispreferably conducted in the presence of HOBT.

Compounds of Formula 1.0, wherein R¹ is a substituent (a), (b), (c),(d), (e), (g), (i), (j), (k), (l), (z.1) or (z.2) can be made byreacting a compound of Formula 10.1 or 30.0 with R¹ -L, wherein L is aleaving group such as Cl, Br, I, or a carboxylate (an anhydride). Thereaction is out in the presence of a base, preferably a tetiary aminesuch as triethylamine or N-methyl morpholine.

Compounds of Formula 1.0, wherein R¹ is a substituent (m) to (q) can bemade by reacting a compound of Formula 10.1 or 30.0 with a pyridylisocyanate, pyridyl N-oxide isocyanate or piperidyl isocyanatecorresponding to the pyridyl, pyridyl N-oxide or piperidyl moiety,respectively, of the substituent groups (m) to (q). The reaction iscarried out in a suitable solvent such as DMF, THF or chloroform usingtechniques well known in the art. Alternatively, these ureas can beprepared by reacting a compound of Formula 10.1 or 30.0 with phosgene toform a chloroformate intermediate (R¹ is --(O)Cl). This chloroformate isgenerally not isolated, and is reacted with pyridyl amine, pyridylN-oxide amine or piperidyl amine corresponding to the pyridyl, pyridylN-oxide or piperidyl moiety, respectively, of the substituent groups (m)to (q) by techniques well known in the art.

Compounds of Formula 1.0 wherein R¹ is a substituent (r) to (v) can bemade by reacting a compound of Formula 10.1 or 30.0 with a pyridylchloroformate or piperidyl chloroformate; or, alternatively, reacting acompounds of Formulas 10.1 or 30.0 with excess phosgene and reacting thechloroformate thus produced with a hydroxypyridyl N-oxide. The reactionis carried out in a suitable solvent, such as dichloromethane, in thepresence of a tertiary amine, such as pyridine, by techniques well knownin the art.

Reductive alkylation of the compound of Formula 10.1 or 30.0 isaccomplished by reacting the compound of Formula 10.1 or 30.0 with analdehyde in DMF with a dehydrating agent such as molecular sieves atroom temperature (about 25° C.). This reaction is followed by reductionof the intermediate imine with a reducing agent such as sodiumcyanoborohydride or sodium triacetoxyborohydride. The reduction isusually carried out at room temperature in a suitable solvent such asDMF.

When compounds of Formulas 10.1 (X is N) or 30.0 (X is CH) are acylatedto make the compounds of Formula 1.0 wherein R¹ is substituents (e) or(g), the protected compounds of Formulas 32.0 and 33.0, respectively areformed (CPh₃ represents triphenylmethyl). These protected compounds canbe deprotected by using trifluoroacetic acid and triethylsilane to yieldthe compounds of Formulas 1.2 and 1.3, respectively. The compounds ofFormulas 1.2 and 1.3 are isolated as the hydrochloride salt followingthe procedure described in Example 1E WO95/00497. ##STR36##

When compounds of Formulas 10.1 (X is N) or 30.0 (X is CH) arereductively alkylated to make the compounds of Formula 1.0 wherein R¹ issubstituents (f) or (h), the protected compounds of Formulas 34.0 and35.0, respectively am formed. These protected compounds can bedeprotected by using trifluoroacetic acid and triethylsilane to yieldthe compounds of Formulas 1.4 and 1.5, respectively. ##STR37##

Certain compounds of Formula (1.0) can be convened to other compounds ofthe Formula (1.0) using standard reaction conditions. For example,compounds of the formula (1.0) wherein R² is --CO₂ H, (i.e., --C(O)OR⁸and R⁸ is H), can be prepared by ozonolysis of a compound of Formula(1.0) wherein R² is CH₂ ═CH--, followed by oxidation of the resultingaldehyde.

Compounds of the Formula (1.0) wherein R² is --C(O)OR⁸, where R⁸ isother than H, can be prepared from a compound of the formula (1.0)wherein R² is --CO₂ H by treating with SOCl₂ or oxalyl chloride, thenwith an alcohol of the formula R⁸ OH, wherein R⁸ is as defined above.Similarly, compounds of formula (1.0) wherein R² is --C(O)NR⁸ R⁹ can beprepared from a compound of the formula (1.0) wherein R² is --CO₂ H viaessentially the same method but substituting and amine of the formula R⁸R⁹ NH for the alcohol R⁸ OH. Alternatively, compounds of Formula (1.0)wherein R² is --C(O)NR⁸ R⁹ can be prepared by reacting a compound of theFormula (1.0) wherein R² is --CO₂ H with an amine R⁸ R⁹ NH in thepresence of a coupling agent, such as DCC or DEC.

In an analogous manner, compounds of Formula (1.0) wherein R² is alkylsubstituted by a group of the formula --C(O)OR⁸ or --C(O)NR⁸ R⁹ can beprepared via substantially the same procedures as described above toform compounds wherein R is --CO₂ H, --C(O)OR⁸ or --C(O)NR⁸ R⁹, byreplacing the compound of Formula (1.0) wherein R² is CH₂ ═CH-- with anappropriate alkenyl group, (i.e., a group of the formula --(CH₂)_(p)--CH═CH₂, wherein p is 1, 2, 3, 4, etc.).

Compounds of the Formula (1.0) wherein R² contains a substituent offormula --S(O)_(t) R⁸, wherein t=1 or 2, can be prepared by oxidation ofan analogous compound of the formula (1.0) wherein R² contains asubstituent of formula --S(O)_(t) R⁸, wherein t=0, using a suitableoxiding agent, such as a peracid, preferably MCPBA.

In the above processes, it is sometimes desirable and/or necessary toprotect certain R¹, R², etc. groups during the reactions. Conventionalprotecting groups are operable as described in Greene, T. W.,"Protective Groups In Organic Synthesis," John Wiley & Sons, New York,1981, the disclosure of which is incorporated herein by referencethereto. For example, the groups listed in column 1 of Table 1 may beprotected as indicated in column 2 of the table:

                  TABLE 1                                                         ______________________________________                                        PROTECTED GROUP                                                               GROUP TO BE                                                                   PROTECTED       2. PROTECTED GROUP                                            ______________________________________                                        COOH                                                                                           ##STR38##                                                     ##STR39##                                                                                     ##STR40##                                                     ##STR41##                                                                                     ##STR42##                                                    OH                                                                                             ##STR43##                                                    NHR, wherein R is any substituent of an amino group within the scope of       the claims                                                                                     ##STR44##                                                    NH.sub.2                                                                                       ##STR45##                                                    ______________________________________                                    

Compounds useful in this invention are exemplified by the followingexamples, which should not be construed as limiting the scope of thedisclosure. Alternative mechanistic pathways and analogous structureswithin the scope of the invention may be apparent to those skilled inthe art.

PREPARATIVE EXAMPLE 13-(1.1-DIMETHYL-1-ETHYL)-8-CHLORO-5,6-DIHYDRO-11H-BENZO(5.6)CYCLOHEPTA(1,2-b)PYRIDIN-11-ONE##STR46##

To a mixture of 20.05 grams (82.28 mmol) of8-chloro-5,6-dihydro-11H-benzo 5,6!cyclohepta 1,2-b!pyridin-11-one in400 ml of dry THF at -72° C. and under an atmosphere of nitrogen wasadded dropwise over 40 minutes 66.0 ml of 2.7M t-butyl magnesiumchloride in THF. The reaction mixture was slowly warmed to roomtemperature and stirred overnight. The mixture was then poured into 10%aqueous ammonium chloride and extracted four times with CH₂ Cl₂. Thecombined organic portions were dried over MgSO₄, filtered, andconcentrated in vacuo to give the title compound, along with8-chloro-11-(1,1-dimethyl-1-ethyl)-6,11-dihydro-5H-benzo 5,6!cyclohepta1,2-b!pyridin-11-ol. These compounds were separated via flashchromatography to give the title compound, which was recrystallized fromisopropyl ether to give 4.37 grams (18%) of the title compound as awhite solid.

PREPARATIVE EXAMPLE 2

A. 8-CHLORO-6,11-DIHYDRO-11-HYDROXY-5H-BENZO 5.6!-CYCLOHEPTA1.2-b!PYRIDINE ##STR47##

To a mixture of 25.03 g (103 mmol) of 8-chloro-5,6-dihydro-11H-benzo5,6!cyclohepta 1,2-b!pyridin-11-one in 200 mL of methanol at roomtemperature and under a nitrogen atmosphere was added portionwise over aperiod of about 1 hour 4.82 g (124 mmol) of sodium borohydride.Occasional cooling with an ice bath was necessary at times during theaddition in order to avoid excessive reflux. After 1.6 hours the mixturewas poured into ice cold water and then extracted with ethyl acetate(3X). The combined organic portions were washed with brine, dried overMgSO₄, filtered, and concentrated in vacuo. The residue wasrecrystallized from hot isopropyl ether. The remaining filtrate waspurified via flash chromatography (20% ethyl acetate in hexanes) toyield more product which solidified on standing. Both batches werecombined to yield 20.41 g of the title compound as a white solid.

B. 8,11-DICHLORO-6,11-DIHYDRO-5H-BENZO 5,6!CYCLOHEPTA 1.2-b!PYRIDINE##STR48##

To a mixture of 13.3 g (54 mmol) of8-chloro-6,11-dihydro-11-hydroxy-5H-benzo 5,6!cyclohepta 1,2-b!pyridinein 290 mL of toluene at -15° C. and under an atmosphere of nitrogen wasadded via syringe pump over a period of 1 hour 6.20 mL (85.7 mmol) ofthionyl chloride. The extent of reaction was monitored by TLC (50% ethylacetate in hexanes). When completed the mixture was poured into 300 mLof 1.0N aqueous NaOH and extracted with ethyl acetate (5X). The combinedorganic portions were washed with brine, dried over sodium sulfate,filtered, and concentrated in vacuo. The residue was taken up in ethylacetate, quickly filtered through basic alumina, and concentrated againto yield a product which was triturated with pentane to yield 10.22 g ofthe title compound as a tan solid.

PREPARATIVE EXAMPLE 3

A. ETHYL 3-PYRIDYLACETIC ACID 1-N-OXIDE ##STR49##

Ethyl 3-pyridylacetic acid (10 grams) (60.6 mmoles) was dissolved in dryCH₂ Cl₂ (120 ml) and the solution was stirred at -18° C. for 30 minutes.MCPBA (31.34 grams) (181.6 mmoles) was added and the mixture was stirredat -18° C. for 1 hour and then at 25° C. for 87 hours. The reactionmixture was diluted with CH₂ Cl₂ and washed with saturated aqueoussodium bicarbonate and then water. The CH₂ Cl₂ was then dried (magnesiumsulphate), filtered and evaporated to dryness. The residue waschromatographed on silica gel using 3% (10% concentrated ammoniumhydroxide in methanol)-CH₂ Cl₂ as the eluant to give the title compound(Yield: 8.45 grams, 77%, MH⁺ 182).

B. 3-PYRIDYLACETIC ACID 1-N-OXIDE ##STR50##

Ethyl-3-pyridylacetic acid 1-N-oxide (0.2747 grams) (1.5 mmoles) wasdissolved in ethanol (200 proof) (1.22 ml.) and a 1M solution of LiOH inwater (3.64 ml.) (3.0 mmoles) was added and the mixture was stirred at25° C. for 4 hours. 1N HCl (4.28 ml.) was added and the mixture waspumped down to dryness on a rotary evaporator to give the title compound(Yield: 0.2931 grams, 100%).

PREPARATIVE EXAMPLE 4 4-ETHOXYCARBONYLAMINOPYRIDINE ##STR51##

4-Aminopyridine (17.34 grams) (184.3) was dissolved in dry pyridine (217ml.) and cooled to 0° C. over 30 minutes. Ethyl chloroformate (17.2 ml.)(180.7 mmoles) was added and the solution was stirred at 0° C. for 1hour and then at 25° C. for 40 hours. The mixture was diluted with CH₂Cl₂ and washed with saturated aqueous NaHCO₃ and water. The CH₂ Cl₂ wasdried (MgSO₄), filtered and evaporated to dryness. The residue waschromatographed on silica gel using 2%(10% saturated NH₄ OH inMeOH)--CH₂ Cl₂ to give the title compound (Yield: 10 grams, 33%, M⁺166).

By using essentially the same procedure, with the exception that##STR52## was used instead of 4-aminopyridine, the compound ##STR53##was obtained, respectively.

PREPARATIVE EXAMPLE 5

A. PIPERIDINE-4-ACETIC ACID ##STR54##

4-Pyridylacetic acid (7 grams) (40.4 mmoles) was hydrogenated in water(100 ml) using 10% Pd-C at 40 psi at 25° C. for 24 hours. The catalystwas filtered off and washed with water. The aqueous solution was shakenwith BioRad AG1×8 resin (OH⁻ form) (23 ml bed) and after 5 minutes theresin was filtered off and washed with water. The aqueous solution wasevaporated to give the title compound (Yield: 5.2 grams, MH⁺ 144).

B. 1-N-ACETYL-4-PIPERIDINYLACETIC ACID ##STR55##

4-Piperidinylacetic acid (5 grams) (35.0 mmoles) was reacted with aceticanhydride (10.7 grams) (105.0 mmoles) in MeOH (100 ml.) and the mixturewas stirred at 25° C. for 24 hours. The mixture was evaporated todryness and the residue was azeotroped with toluene to give the titlecompound (Yield: 6.4 grams, 99%, MH⁺ 185).

C. 1-N-METHYL-4-PIPERIDINYLACETIC ACID ##STR56##

4-Piperidinylacetic acid (4 grams) (28.0 mmoles) from PreparativeExample 5A was dissolved in water (50 ml) and 37% formalin (2.72 ml)(33.6 mmoles) was added. The mixture was hydrogenated over 10% Pd-C t 55psi at 25° C. for 68 hours. The catalyst was filtered off and washedwith water. The combined filtrates were evaporated to dryness to givethe title compound (MH+158).

D. 1-N-tert-BUTOXYCARBONYLPlPERIDINYL-4-ACETIC ACID ##STR57##

4-Piperidinylacetic acid (41.24 grams) (288.4 mmoles) from PreparativeExample 5A was dissolved in THF-water (1:1) (400 ml) anddi-tert-butyldicarbonate (69.14 grams) (317.3 mmoles) and NaOH (11.52grams) (288.4 mmoles) were added. The mixture was stirred at 25° C. forhours. The solution was then eluted through a bed of washed BioRad 50WX4(RSO3H resin) (150 ml bed) and the resin was eluted with a 1:1 mixtureof THF and water. The eluate was evaporated to dryness to give the titlecompound (Yield: 53.0 grams, 76%).

PREPARATIVE EXAMPLE 6

A. 3-PIPERIDINYLACETIC ACID ##STR58##

3-Pyridylacetic acid hydrochloride (13 grams) (74.9 mmoles) washydrogenated as described in Preparative Example 5A to give a mixture ofunreacted 3-pyridylacetic acid and the title compound (76:24) (8.63grams, MH⁺ 144).

B. 1-N-ACETYL-3-PIPERIDINYLACETIC ACID ##STR59##

The mixture of compounds from Preparative Example 6A (8.56 grams) werereacted with acetic anhydride (8.56 grams) as described in PreparativeExample 5B and the crude mixture of products was diluted in methanol (60ml) and passed over a bed of BioRad AG50WX4 resin (RSO₃ H) and thelatter was eluted with methanol. The eluates were evaporated to drynessto give the title compound (Yield: 1.23 grams, MH⁺ 186).

C. 1-N-METHYL-3-PIPERIDINYLACETIC ACID ##STR60##

The mixture of compounds from Preparative Example 6A (4 grams) and 37%formalin (2.72 ml.) were hydrogenated as described in PreparativeExample 5C to give the title compound (MH⁺ 158).

PREPARATIVE EXAMPLE 7 3-BROMO-8-CHLORO-5,6-DIHYDRO-11H-BENZO5.6!-CYCLOHEPTA 1.2-b!PYRIDIN-11-ONE ##STR61##

Cyclize 3- 2-(3-chlorophenyl)ethyl!-4-bromo-2-pyridine carbonitrile(10.7 g, 32.8 mmol) in triflic acid (82 mL) at 60° C. for 2 hours andthen at room temperature for 2 hours. Add 80 mL of 5N HCl carefully,then reflux in an oil bath (120° C.) for 30 minutes. Cool the solutionand pour into ice and basify with 25% NaOH solution. Extract the productwith CH₂ Cl₂ and wash with brine. Dry the organic layer with Na₂ SO₄,filter and remove the solvent to give crude product (10.4 g). Purify thecrude product with flash chromatography on silica gel and elute with 15%ethyl acetate-hexane to give the title compound as a white solid (9 g,27.95 mmol, Yield 85.2% MH⁺ 322).

PREPARATIVE EXAMPLE 8 3-PYRIDYLISOCYANATE, HYDROCHLORIDE ##STR62##

A 1.93 solution of phosgene in toluene (20%) (584 mL) was diluted withdry CH₂ Cl₂ (1 L) and the mixture was stirred at 0° C. under nitrogenatmosphere. A solution of 3-aminopyridine (21.1 grams) and dry pyridine(19 mL) dissolved in dry CH₂ Cl₂ (600 mL) was added dropwise to thestirred solution at 0° C. over a period of 5.5 hours. The mixture wasstirred at 0°-25° C. for an additional 48 hours. A stream of nitrogenwas passed through the solution to remove most of the phosgene and thesolution was then evaporated until almost all of the solvent was removedto give the title compound which was then taken up in dry pyridine (850mL) to give a stock solution of the title compound.

PREPARATIVE EXAMPLE 9 ##STR63##

The preparation of the starting material for this reaction was describedin The Journal of Organic Chemistry, 1990, 55, pp. 3341-3350 byPiwinski, J. J., et al. By substituting in Preparative Example 2A,8-chloro-11H-benzo 5,6!cyclo-hepta 1,2-b!pyridin-11 -one (11.53 g)(47.71 mmoles) for 8-chloro-5,6-dihydro-11H-benzo 5,6!cyclohepta1,2-b!pyridin-11-one and employing basically the same methods as steps Aand B of Preparative Example 2, one obtains 11.53 g (36%) of the titlecompound (MH⁺ 312).

PREPARATIVE EXAMPLE 10 ##STR64##

By substituting in Preparative Example 2A, 3-bromo-8-chloro-11-(1-15piperazinyl) -5,6-dihydro- 11H-benzo 5,6!cyclohepta 1,2-b!pyridin-11-one(1.5 g, 4.65 mmoles) (Preparative Example 7A) for8-chloro-5,6-dihydro-11H-benzo 5,6!cyclohepta 1,2-b!pyridin-11-one andusing the same methods as described in steps A and B of PreparativeExample 2, one obtains the title compound (1.31 g, 72%, MH⁺ 392).

PREPARATIVE EXAMPLE 11 ##STR65##

Combine 10 g (60.5 mmol) of ethyl 4-pyridylacetate and 120 mL of dry CH₂Cl₂ at -20° C., add 10.45 g (60.5 mmol) of MCPBA and stir at -20° C. for1 hour and then at 25° C. for 67 hours. Add an additional 3.48 g (20.2mmoles) of MCPBA and stir at 25° C. for 24 hours. Dilute with CH₂ Cl₂and wash with saturated NaHCO₃ (aqueous) and then water. Dry over MgSO₄,concentrate in vacuo to a residue, and chromatograph (silica gel,2%-5.5% (10% NH₄ OH in MeOH)/CH₂ Cl₂) to give 8.12 g of the productcompound (Et represents --C₂ H₅ in the formula). Mass Spec.: MH⁺ =182.15##STR66##

Combine 3.5 g (19.3 mmol) of the product of Step A, 17.5 mL of ethanoland 96.6 mL of 10% NaOH (aqueous) and heat the mixture at 67° C. for 2hours. Add 2N HCl (aqueous) to adjust to pH=2.37 and concentrate invacuo to a residue. Add 200 mL of dry ethanol, filter through celite®and wash the filter cake with dry EtOH (2×50 ml).

Concentrate the combined filtrates in vacuo to give 2.43 g of the titlecompound.

PREPARATIVE EXAMPLE 12 ##STR67##

Combine 10 g (65.7 mmol) of 3-methoxycarbonylaminopyridine and 150 mL ofCH₂ Cl₂, cool to 0° C. and slowly add (dropwise) a solution of 13.61 g(78.84 mmol) of MCPBA in 120 mL of CH₂ Cl₂ at 0° C. over a period of 1hour. Stir the mixture at 25° C. for 5 days, then wash with saturatedNaHCO₃ (aqueous), then water and dry over MgSO₄. Concentrate in vacuo toa residue and chromatograph (silica gel, 2%-5% (10% NH₄ OH in MeOH)/CH₂Cl₂) to give the product compound. Mass Spec.: MH⁺ =169

PREPARATIVE EXAMPLE 13 ##STR68##

Combine 5 g (36.0 mmol) of isonicotinic acid 1-N-oxide and 150 mL ofanhydrous DMF, add 5.5 mL (39.6 mmol) of triethylamine and stir at 0° C.for 0.5 hours. Slowly add (dropwise) 8.5 mL (39.6 mmol) ofdiphenylphosphoryl azide at 0° C. over 10 minutes, stir at 0° C. for 1hour and then at 25° C. for 24 hours (as generally described in Pavia,et al., Journal of Medicinal Chemistry, 33, 854-861 (1990). Concentratein vacuo to a residue and chromatograph (silica gel, 0.5%-1% MeOH/CH₂Cl₂) to give 5.9 g of the product compound.

Using nicotinic acid 1-N-oxide and substantially the same procedure asdescribed for Preparative Example 13 the following compound wasprepared: ##STR69##

PREPARATIVE EXAMPLE 15 ##STR70##

Hydrogenate 25 g (144 mmol) of 3-pyridylacetic acid hydrochloride for144 hours using the procedure described in Preparative Example 5A togive 20 g of the product compound. Mass Spec.: MH⁺ =144. ##STR71##

React 12 g (83.8 mmol) of the product of Step B for 148 hours using theprocedure described in Preparative Example 5D, to give 17.5 g of theproduct compound. Mass Spec.: MH⁺ =244.25

PREPARATIVE EXAMPLE 15 ##STR72##

Combine 25 g (164.4 mmol) of methyl 3-pyridylcarbamate and 163.3 mL of1N HCl (aqueous), stir until all of the solid dissolves, thenhydrogenate over 10% Pd/C at 25° C. at 55 psi for 220 hours. Filter,wash the solids with water and treat the combined filtrates with 150 mLof BioRad AG1X8 ion exchange resin (OH⁻). Filter, wash the resin withwater and concentrate the filtrate to a volume of 100 mL. Add 16.43 mL(197.3 mmol) of 37% formalin and hydrogenate over 10% Pd/C at 25° C. at55 psi for 89 hours. Filter, wash the solids with water and concentratein vacuo to give 24.3 g of the title compound. Mass Spec.: MH⁺ =173.2

PREPARATIVE EXAMPLE 16 ##STR73##

Cool 50.0 g (20.5 mmol) of 8-chloro-5,6-dihydro-11H-benzo 5,6!cyclohepta1,2-b!pyridin-11-one to 0° C., slowly add 75 mL (93.69 mmol) of sulfurmonochloride over 20 minutes, then slowly add 25 mL (48.59 mmol) of Br₂over 15. Heat at 95° C. for 20 hour, add 12.5 mL (24.3 mmol) of Br₂ andheat for a another 24 hours. Cool the mixture, and slowly add to amixture of CH₂ Cl₂ and 1N NaOH (aqueous) at 0° C. Wash the organic phasewith water, dry over MgSO₄ and concentrate in vacuo to a residue.Chromatograph the residue (silica gel, 500 mL CH₂ Cl₂ then 0.2%-5% (10%NH₄ OH in MeOH)/CH₂ Cl₂), then chromatograph again (silica gel, 3%-8.5%EtOAc/hexane) to give 8.66 g of the product compound. Mass Spec.: MH⁺=322

PREPARATIVE EXAMPLE 17 ##STR74##

Combine 10 mL of dry CH₂ Cl₂ and 914.6 mL (28.1 mmol) of a 1.93Msolution of phosgene in toluene, cool to 0° C. and slowly add (dropwise)a solution of 0.6484 g (5.62 mmol) of 4-hydroxy-1-N-methylpiperidine,1.214 mL (15 mmol) of pyridine and 10 mL of dry CH₂ Cl₂ over 10 minutes,then stir at 0° to 25° C. for 2 hours. Purge excess phosgene with N₂then concentrate in vacuo to give the title compound.

EXAMPLE 1 ##STR75##

Following the procedure of Villani et al., J. Med. Chem. 15, 750 (1972),the product of Preparative Example 2 was dissolved in acetic acid andexcess zinc was added. This mixture was heated for two hours at 80° C.The reaction mixture was filtered and concentrated under vacuo. Aqueoussodium bicarbonate was added and the mixture was extracted with ethylacetate. The organic layer was washed with water and dried overmagnesium sulfate, filtered and concentrated under vacuo. Theconcentrated material was chromatographed on silica gel using ethylacetate-hexane to obtain the product. ##STR76##

Piperazine protected with a BOC group (commercially available) wasdissolved in methylene chloride and 1.2 equivalents ofcarbonyldiimidazole was added at 0° C. and the mixture was stirred for15 minutes. Sodium chloride solution was added and the mixture wasextracted with methylene chloride. The organic layer was dried overmagnesium sulfate, filtered and concentrated under vacuo to obtain theproduct. ##STR77##

The product of Step A was dissolved in tetrahydrofuran and cooled to-78° C. One equivalent of butyl lithium was added and the mixture wasstirred for 10 minutes. One equivalent of the product of Step B intetrahydrofuran was added and the mixture was stirred for 1 hour at -78°C., and then at 25° C. for 18 hours. Water was added and the mixture wasextracted with ethyl acetate, the organic layer was dried over magnesiumsulfate and concentrated under vacuo. The concentrated material waschromatographed on silica gel using ethyl acetate-hexane to give theproduct as a tan solid, M+1=442. ##STR78##

The product of Step C was dissolved in HCl-Dioxane and stirred untilreaction was completed (about 1 hour). Concentration in vacuo gave theproduct. ##STR79##

The product of Step D was dissolved in N,N-dimethylformamide and the pHwas adjusted to 6 with triethylamine. Sodium triacetoxyborohydride, 1.25equivalents, and crushed 4A molecular sieves were added to the solution.The resulting mixture was cooled to 0° C. under nitrogen and 1.5equivalents of the reactant aldehyde (see Example 1 on page 45 ofWO95/00497) in N,N-dimethylformamide was added dropwise. After additionwas completed, the mixture was stirred at 0° C. for 21/2 hours. Themixture was then diluted with ethyl acetate, filtered, and washed withsodium bicarbonate solution. The organic layer was dried over magnesiumsulfate, filtered and concentrated under vacuo. The concentratedmaterial was chromatographed on silica gel using ethyl acetate-hexane togive a white solid. M+1 =772. ##STR80##

The product of Step E was treated with 1N HCl in acetic acid at roomtemperature for 1/2 hour, then at 47° C. for 15 minutes, cooled to 20°C. and treated with triethylsilane for 1/2 hour. The hydrochloride ofthe title compound was isolated as a white powder by diluting thereaction mixture with ethyl acetate followed by centrifugation. M+1=431.

EXAMPLE 2

If one were to follow the procedures described in Steps A and B then onewould obtain a compound of the formula: ##STR81##

Dissolve the product of Example 1, Step D, in N,N-dimethylformamide andadd 1 equivalent of the reactant carboxylic acid (commerciallyavailable), 1 equivalent of 1-hydroxybenzotriazole, 1 equivalent of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (DEC) and 1equivalent of triethylamine. Stir until reaction is complete, about 18hours. Concentrate in vacuo. Chromatograph on silica gel using ethylacetate-hexane to obtain the product. ##STR82##

React and purify as in Example 1, Step F, to obtain the product.

EXAMPLE 3

If one were to follow the procedures described in Steps A to F then onewould obtain a compound of the formula: ##STR83##

Follow the procedure set forth in Example 1, Step A, using the productof Preparative Example 10 to obtain the product. ##STR84##

Follow the procedure of Example 1, Step B, using the product of Example3, Step B, to obtain the product. ##STR85##

Follow the procedure of Example 1, Step C, using the products of Example3, Steps A and B, to obtain the product. ##STR86##

Follow the procedure of Example 1, Step D, using the product of Example3, Step C, to obtain the product. ##STR87##

Follow the procedure of Example 1, Step E, using the product of Example3, Step D, and the aldehyde to give the product. ##STR88##

Follow the procedure of Example 1, Step F, using the product of Example3, Step E, to give the product.

EXAMPLE 4

If one were to follow the procedures described in Steps A to E then onewould obtain a compound of the formula: ##STR89##

Follow the procedure of Example 1, Step A, using the product ofPreparative Example 9 to obtain the product. ##STR90##

Follow the procedure of Example 1, Step B, to obtain the product.##STR91##

Follow the procedure of Example 1, Step C, using the products of Example4, Steps A and B,to obtain the product. ##STR92##

Follow the procedure of Example 1, Step D, using the product of Example4, Step C, to obtain the product. ##STR93##

Follow the procedure of Example 2, Step A, using the product of Example4, Step D, and Preparative Example 11, to obtain the product.

EXAMPLE 5

If one were to follow the procedures described in Steps A to F then onewould obtain a compound of the formula: ##STR94##

Dibenzosuberane was dissolved in tetrahydrofuran and cooled to 0° C.under nitrogen. 1.5 Equivalents of n-butyl lithium was added and allowedto warm to 20° C., and was kept at 20° C. for 1 hour. The reactionmixture was poured onto crushed solid carbon dioxide. After 0.5 hours10% aqueous hydrochloric acid was added and the mixture was extractedwith methylene chloride. The organic layer was extracted with 0.1Msodium hydroxide. The aqueous layer was cooled and the pH was adjustedto 2 with 12M hydrochloric acid. The precipitated product was filteredand dried giving a white solid. ##STR95##

Make the piperazine reactant according to the procedure in Example 13 ofWO95/00497. Follow the procedure of Example 2, Step A, above, to obtainthe product. ##STR96##

Dissolve the product of Step B in dry, degassed N,N-dimethylforamide andcool to 0° C. Add 1.3 equivalents of sodium hydride followed by 1.4equivalents of 3-chloromethylpyridine. After 3 hours quench the reactionwith saturated ammonium chloride solution. Concentrate under vacuo andpartition between ethyl acetate and sodium bicarbonate solution. Dry theorganic layer over magnesium sulfate, filter and concentrate undervacuo. Chromatograph the residue on silica gel using ethylacetate-hexane. ##STR97##

Follow the procedure of Example 1, Step D, to obtain the product.##STR98##

Prepare the reactant aldehyde by procedures similar to those describedin Example 1 of WO95/00497. Follow the procedure of Example 1, Step E,above to obtain the product. ##STR99##

Follow the procedure of Example 1, Step F, to obtain the product.

EXAMPLES 6-10 ##STR100##

React the title compound of Example 13A of WO 95/00497 withbenzyloxycarbonyl chloride according to standard conditions well knownin the art, to obtain the N-Cbz (benzyloxycarbonyl) protected alcoholshown above. Purification of the protected alcohol, according toprocedures well known in the art, and then reaction of the protectedalcohol with the reagents in Column 1 of Table 2 would give thecorresponding N-Cbz protected intermediates having R as defined inColumn 2 of Table 2. After purification according to techniques wellknown in the art, the protected intermediate may be selectivelydeprotected (to remove the Cbz group) using mild catalytic hydrogenationprocedures well known in the art. Following deprotection, purificationby known techniques would yield the BOC-protected intermediate havingthe R group shown in Column 2 of Table 2.

                  TABLE 2                                                         ______________________________________                                        Column 1 - (Reagents)                                                                        Column 2 - R Group                                             ______________________________________                                                       Example 6                                                       ##STR101##                                                                                   ##STR102##                                                                   Prepare as described in Example 14 of                                         WO 95/00497.                                                   C.sub.6 H.sub.5 SSC.sub.6 H.sub.5 + (n-Bu).sub.3 P                                           Example 7                                                                      ##STR103##                                                                   Prepare as described in Examples 20B                                          and 20C of WO 95/00497.                                         ##STR104##                                                                                   ##STR105##                                                     ##STR106##                                                                                   ##STR107##                                                    n-C.sub.3 H.sub.7 I + NaH                                                                    Example 10                                                                    n-C.sub.3 H.sub.7 O                                                           Prepare as described in Example 13C                                           of WO 95/00497.                                                ______________________________________                                    

EXAMPLE 11 ##STR108##

Convert the title compound from Example 27D of WO 95/00497, by thescheme shown above, using procedures well known in the art, into1-tert-butoxycarbonyl-2(S)-(4-acetylaminobutyl)piperazine.

EXAMPLE 12

If one were to follow the procedures in Steps A to G, then one wouldobtain the compound: ##STR109##

Prepare the starting material according to the procedure described inExample 4C of WO89/10369. Convert the starting material into the chloroproduct by the method described in U.S. Pat. No. 3,409,621. ##STR110##

Follow the procedure in Example 1, Step A, to obtain the product.##STR111##

React the product of Step A with the product of Example 7 by the methodof Example 1, Step B, to obtain the product. ##STR112##

Follow the procedure in Example 1, Step C, to obtain the product.##STR113##

Follow the procedure in Example 1, Step D, to obtain the product.##STR114##

React the product of Example 12, Step E, with the BOC protected4-piperidinylacetic acid of Preparative Example 5D, according to theprocure in Example 2, Step A, to obtain the product. ##STR115##

Dissolve the product of Step F in HCl-Dioxane and stir for 1 hour.Concentrate in vacuo. Partition between sodium bicarbonate solution andethyl acetate. Dry the organic layer over magnesium sulfate, filter andconcentrate under vacuo. Dissolve the residue in methylene chloride andadd excess trimethylsilylisocyanate. Stir under nitrogen for 18 hours.Add additional trimethylsilylisocyanate and stir until the reaction iscomplete. Wash with aqueous sodium bicarbonate solution. Dry the organiclayer over magnesium sulfate, filter and concentrate in vacuo.Chromatograph the residue on silica gel using methanol-methylenechloride to give the product.

EXAMPLE 13

If one were to follow the procedures in Steps A to E, then one wouldobtain the compound: ##STR116##

According to the procedure of D. L. Comins, et al., in Tat. Lett., 4549(1986), dissolve 4-methoxypyridine in THF and cool to -23° C. Addbenzylchloroformate dropwise (1 equivalent) followed by 1 equivalent ofbutyl magnesium chloride in THF added dropwise. Pour into 10%hydrochloric acid and extract with ether. Dry over MgSO₄ andconcentrate. (Ph in the above formula represents phenyl). ##STR117##

Dissolve the product of Step A in ethanol containing 10% palladium oncarbon and hydrogenate at 60 psi. Filter and concentrate under vacuo toobtain the product. ##STR118##

Dissolve the product of Step B in tetrahydrofuran, cool to 0° C. undernitrogen and add one equivalent of sodium hydride. After stirring for 15minutes, one equivalent of methyl iodide is added. Stir the reaction for15 minutes, concentrate under vacuo and chromatograph on silica gelusing methanol-methylene chloride. ##STR119##

Dissolve the product of Step C in ethanol and add an excess of sodiumborohydride. Concentrate under vacuo. Partition between water and ethylacetate. Dry the organic layer over magnesium sulfate, filter andconcentrate under vacuo. ##STR120##

Dissolve the product of Step D in pyridine containing an excess ofthionyl chloride. Stir for 18 hours and concentrate in vacuo. Partitionbetween ethyl acetate and aqueous sodium bicarbonate. Dry the organiclayer over magnesium sulfate, filter and concentrate in vacuo to obtainthe product.

EXAMPLE 14

If one were to follow the procedure of Steps A to F, then one wouldobtain the compound: ##STR121##

5-chlorodibenzosuberane, 48.18 g (0.2 mole), was dissolved in 400 mL oftoluene. Silver cyanide, 36.7 g (0.27 mole), was added and the mixturewas refluxed for 24 hours. The mixture was cooled, filtered andconcentrated in vacuo. The residue was recrystallized from 2-propylether and hexane to give 38.8 g of the product. MP=94.2°-94.9° C.##STR122##

Dissolve the product of Example 12, Step E, in THF and add oneequivalent of magnesium. Stir until all of the magnesium has reacted.Add this solution dropwise to a solution of one equivalent of theproduct of Step A in THF. Stir for 1 hour then quench with aqueousammonium chloride solution. Extract with ethyl acetate. Dry the organiclayer over magnesium sulfate, filter and concentrate under vacuo.Chromatograph on silica gel using methanol-methylene chloride to obtainthe product. ##STR123##

Dissolve the product of Step B in dry toluene containing 2 equivalentsof triethylamine. Warm to 80° C. and add 9 equivalents of ethylchloroformate. Stir at 80° C. until the reaction is complete, about 2hours. Filter and concentrate under vacuo. Chromatograph on silica gelusing ethyl acetate-hexane to obtain the product. ##STR124##

Dissolve the product of Step C in 12M hydrochloric acid and reflux untilcomplete, about 6 hours. Adjust the pH to 8 with solid sodium hydroxideand filter the precipitated product. Chromatograph on silica gel usingmethanol-methylene chloride and ammonium hydroxide to give the product.##STR125##

Follow the procedure in Example 1, Step E, to obtain the product.##STR126##

Follow the procedure in Example 1, Step F, to obtain the product.

EXAMPLE 15 ##STR127##

The product of Example 1D was dissolved in DMF and cooled to 0° C. undernitrogen. To this solution was added 2 equivalents of 4-pyridyl aceticacid, 6 equivalents of triethylamine, 2 equivalents of1-hydroxybenzotriazole (HOBT), and 2 equivalents of DEC. The reactionmixture was stirred at 0° C. overnight. Then the reaction mixture wasdiluted with aqueous sodium bicarbonate solution and extracted withethyl acetate. The organic layer was concentrated under vacuo and theresidue was chromatographed on silica gel using methanol-methylenechloride as the solvent. Fractions containing the product wereconcentrated under vacuo to give the title compound as a white foam.M+1=461.

EXAMPLE 16

If one were to follow the procedure described below, then one wouldobtain the indicated compound: ##STR128##

Add a solution of iodine in methanol to the product of Example 3, StepF, in methanol until a slight yellow color persists. Concentrate invacuo and chromatograph the residue by HPLC using a C₁₈ column and asolvent of water-acetonitrile and 0. 1% trifluoroacetic acid.Concentrate in vacuo to give the product.

EXAMPLE 17

If one were to follow the procedure described, then one would obtain thecompound: ##STR129##

React the product of Example 3D with the product of Preparative Example17 in dichloromethane in the presence of pyridine at 25° C. for 20 to100 hours to give the title compound.

EXAMPLE 18

If one were to follow the procedure described, then one would obtain thecompound: ##STR130##

Heat he product (acylazine) from Preparative Example 13 under reflux inanhydrous toluene to convert it into the corresponding isocyanate insitu. Add the product of Example 3D in anhydrous toluene to the mixtureand stir this mixture at 25° C. for 20 hours to give the title compound.

ASSAYS

The utility of the compounds of the present invention can bedemonstrated by the following assay procedures.

1. In vitro enzyme assays: Inhibition of farnesyl protein transferaseand geranylgeranyl protein transferase.

Both famesyl protein transferase (FPT) and geranylgeranyl proteintransferase (GGPT) I are partially purified from rat brain by ammoniumsulfate fractionation followed by Q-Sepharose (Pharmacia, Inc.) anionexchange chromatography essentially as described by Yokoyama et al(Yokoyama, K., et al., (1991), A protein geranylgeranyltransferase frombovine brain: Implications for protein prenylation specificity, Proc.Natl. Aced. Sci USA 88:5302-5306, the disclosure of which isincorporated herein by reference thereto). Human famesyl proteintransferase is also expressed in E. coli, using cDNA clones encodingboth the α and β subunits. The methods used are similar to thosepublished (Omer, C. et al., (1993). Characterization of recombinanthuman famesyl protein transferase: Cloning, expression, famesyldiphosphate binding, and functional homology with yeast prenyl-proteintransferases, Biochemistry 32:5167-5176). Human famesyl proteintransferase is partially-purified from the soluble protein fraction ofE. coli as described above. The tricyclic famesyl protein transferaseinhibitors disclosed herein inhibit both human and rat enzyme withsimilar potencies. Two forms of val¹² -Ha-Ras protein are prepared assubstrates for these enzymes, differing in their carboxy terminalsequence. One form terminates in cysteine-valine-leucine-serine(Ras-CVLS) the other in cystein-valine-leucine-leucine (Ras-CVLL).Ras-CVLS is a substrate for the famesyl protein transferase whileRas-CVLL is a substrate for geranylgeranyl protein transferase I. ThecDNAs encoding these proteins are constructed so that the proteinscontain an amino-terminal extension of 6 histidine residues. Bothproteins are expressed in Escherichia coli and purified using metalchelate affinity chromatography. The radiolabelled isoprenylpyrophosphate substrates, ³ H!farnesyl pyrophosphate and ³H!geranylgeranyl pyrophosphate, are purchased from a commercial source,such as DuPont/New England Nuclear.

Several methods for measuring farnesyl protein transferase activity areknown (Reiss et al 1990, Cell 62:81; Schaber et al 1990, J. Biol. Chem.265:14701; Manne et al 1990, PNAS 8.7:7541; and Barbacid & Manne 1993,U.S. Pat. No. 5,185,248). The activity is assayed by measuring thetransfer of ³ H!famesyl from ³ H!farnesyl pyrophosphate to Ras-CVLSusing conditions similar to those described by Reiss et al., 1990 (Cell62:81) The reaction mixture contains 40 mM Hepes, pH 7.5; 20 mMmagnesium chloride; 5 mM dithiothreitol; 0.25 μM ³ H!farnesylpyrophosphate; 10 μl Q-Sepharose-purified farnesyl protein transferase;the indicated concentration of tricyclic compound or dimethylsulfoxide(DMSO) vehicle control (5% DMSO final); and 5 μM Ras-CVLS in a totalvolume of 100 μl. The reaction is allowed to proceed for 30 minutes atroom temperature and then stopped with 0.5 ml of 4% sodium dodecylsulfate (SDS) followed by 0.5 ml of cold 30% trichloracetic acid (TCA).Samples are allowed to sit on ice for 45 minutes and precipitated Rasprotein is then collected on GF/C filter paper mats using a Brandel callharvester. Filter mats are washed once with 6% TCA, 2% SDS andradioactivity is measured in a Wallac 1204 Betaplate BS liquidscintillation counter. Percent inhibition is calculated relative to theDMSO vehicle control.

The geranylgeranyl protein transferase I assay is essentially identicalto the farnesyl protein transferase assay described above, with twoexceptions: ³ H!geranylgeranylpyrophosphate replaces farnesylpyrophosphate as the isoprenoid donor and Ras-CVLL is the proteinacceptor. This is similar to the assay reported by Casey et al (Casey,P. J., et al., (1991), Enzymatic modification of proteins with ageranylgeranyl isoprenoid, Proc. Natl. Aced. Sci, USA 88:8631-8635, thedisclosure of which is incorporated herein by reference).

2. Cell-Based Assay: Transient expression of val¹² -Ha-Ras-CVLS andval¹² -Ha-Ras-CVLL in COS monkey kidney calls: Effect of farnesylprotein transferase inhibitors on Ras processing end on disordered cellgrowth induced by transforming Ras.

COS monkey kidney cells are transfected by electroporation with theplasmid pSV-SPORT (Gibco/BRL) containing a cDNA insert encoding eitherRas-CVLS or Ras-CVLL, leading to transient overexpression of a Rassubstrate for either farnesyl protein transferase or geranylgeranylprotein transferase I, respectively (see above).

Following electroporation, cells are plated into 6-well tissue culturedishes containing 1.5 ml of Dulbecco's-modified Eagle's media (GIBCO,Inc.) supplemented with 10% fetal calf serum and the appropriate famesylprotein transferase inhibitors. After 24 hours, media is removed andfresh media containing the appropriate drugs is re-added.

48 hours after electroporation cells are examined under the microscopeto monitor disordered cell growth induced by transforming Ras. Cellsexpressing transforming Ras become more rounded and refractile andovergrow the monolayer, reminiscent of the transformed phenotype. Cellsare then photographed, washed twice with 1 ml of cold phosphate-bufferedsaline (PBS) and removed from the dish by scraping with a rubberpoliceman into 1 ml of a buffer containing 25 mM Tris, pH 8.0; 1 mMethylenediamine tetraacetic acid; 1 mM phenylmethylsulfonyl fluoride; 50μM leupeptin; and 0.1 μM pepstatin. Cells are lysed by homogenizationand cell debris is removed by centrifugation at 2000×g for 10 min.

Cellular protein is precipitated by addition of ice-cold trichloroaceticacid and redissolved in 100 μl of SDS-electrophoresis sample buffer.Samples (5-10 μl ) are loaded onto 14% polyacrylamide minigels (Novex,Inc.) and electrophoresed until the tracking dye neared the bottom ofthe gel. Proteins resolved on the gels are electroblotted ontonitrocellulose membranes for immunodetection.

Membranes are blocked by incubation overnight at 4° C. in PBS containing2.5% dried milk and 0.5% Tween-20 and then incubated with a Ras-specificmonoclonal antibody, Y13-259 (Furth, M. E., et al., (1982), Monoclonalantibodies to the p21 products of the transforming gene of Harvey murinesarcome virus and of the cellular ras gene family, J. Virol.43:294-304), in PBS containing 1% fetal calf serum for one hour at roomtemperature. After washing, membranes are incubated for one hour at roomtemperature with a 1:5000 dilution of secondary antibody, rabbitanti-rat IgG conjugated to horseradish peroxidase, in PBS containing 1%fetal calf serum. The presence of processed and unprocessed Ras-CVLS orRas-CVLL is detected using a colorimetric peroxidase reagent(4-chloro-1-naphthol) as described by the manufacturer (Bio-Rad).

3. Cell Mat Assay;

Normal human HEPM fibroblasts are planted in 3.5 cm dishes at a densityof 5×10⁴ cells/dish in 2 ml growth medium, and incubated for 3-5d toachieve confluence. Medium is aspirated from each dish and the indicatortumor cells, T24-BAG4 human bladder carcinoma cells expressing anactivated H-ras gene, are planted on top of the fibroblast monolayer ata density of 2×10³ cells/dish in 2 ml growth medium, and allowed toattach overnight. Compound-induced colony inhibition is assayed byaddition of serial dilutions of compound directly to the growth medium24 h after tumor cell planting, and incubating cells for an additional14 d to allow colony formation. Assays are terminated by rinsingmonolayers twice with phosphate-buffered saline (PBS), fixing themonolayers with a 1% glutaraldehyde solution in PBS, then visualizingtumor cells by staining with X-Gal (Price, J., et al., Lineage analysisin the vertebrate nervous system by retrovirus-mediated gene transfer,Proc. Natl. Acad. Sci. 84, 156-160(1987)). In the colony inhibitionassay, compounds are evaluated on the basis of two IC₅₀ values: theconcentration of drug required to prevent the increase in tumor cellnumber by 50% (tlC₅₀) and the concentration of drug required to reducethe density of cells comprising the cell mat by 50% (mlC₅₀). Both lC₅₀values are obtained by determining the density of tumor cells and matcells by visual inspection and enumeration of cells per colony and thenumber of colonies under the microscope. The therapeutic index of thecompound is quantitatively expressed as the ratio of mlC₅₀ /tlC₅₀, withvalues greater than one indicative of tumor target specificity.

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets and suppositories. Thepowders and tablets may be comprised of from about 5 to about 70 percentactive ingredient. Suitable solid carriers are known in the art, e.g.magnesium carbonate, magnesium stearate, talc, sugar, lactose. Tablets,powders, cachets and capsules can be used as solid dosage forms suitablefor oral administration.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides or cocoa butter is first melted, and the activeingredient is dispersed homogeneously therein as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool and thereby solidify.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection.

Liquid form preparations may also include solutions for intranasaladministration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally.The transdermal compositions can take the form of creams, lotions,aerosols and/or emulsions and can be included in a transdermal patch ofthe matrix or reservoir type as are conventional in the art for thispurpose.

Preferably the compound is administered orally.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component, e.g., an effectiveamount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 0.1 mg to 1000 mg, more preferably fromabout 1 mg. to 300 mg, according to the particular application.

The actual dosage employed may be varied depending upon the requirementsof the patient and the severity of the condition being treated.Determination of the proper dosage for a particular situation is withinthe skill of the art. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day if desired.

The amount and frequency of administration of the compounds of theinvention and the pharmaceutically acceptable salts thereof will beregulated according to the judgment of the attending clinicianconsidering such factors as age, condition and size of the patient aswell as severity of the symptoms being treated. A typical recommendeddosage regimen is oral administration of from 10 mg to 2000 mg/daypreferably 10 to 1000 mg/day, in two to four divided doses to blocktumor growth. The compounds are non-toxic when administered within thisdosage range.

The following are examples of pharmaceutical dosage forms which containa compound of the invention. The scope of the invention in itspharmaceutical composition aspect is not to be limited by the examplesprovided.

Pharmaceutical Dosage Form Examples EXAMPLE A

    ______________________________________                                        Tablets                                                                       No.     Ingredients      mg/tablet                                                                              mg/tablet                                   ______________________________________                                        1.      Active compound  100      500                                         2.      Lactose USP      122      113                                         3.      Corn Starch, Food Grade,                                                                        30       40                                                 as a 10% paste in                                                             Purified Water                                                        4.      Corn Starch, Food Grade                                                                         45       40                                         5.      Magnesium Stearate                                                                              3        7                                                  Total            300      700                                         ______________________________________                                    

Method of Manufacture

Mix Item Nos. 1 and 2 in a suitable mixer for 10-15 minutes. Granulatethe mixture with Item No. 3. Mill the damp granules through a coarsescreen (e.g., 1/4", 0.63 cm) if necessary. Dry the damp granules. Screenthe dried granules if necessary and mix with Item No. 4 and mix for10-15 minutes. Add Item No. 5 and mix for 1-3 minutes. Compress themixture to appropriate size and weigh on a suitable tablet machine.

EXAMPLE B

    ______________________________________                                        Capsules                                                                      No.   Ingredient       mg/capsule mg/capsule                                  ______________________________________                                        1.    Active compound  100        500                                         2.    Lactose USP      106        123                                         3.    Corn Starch, Food Grade                                                                         40         70                                         4.    Magnesium Stearate NF                                                                           7          7                                                Total            253        700                                         ______________________________________                                    

Method Of Manufacture

Mix Item Nos. 1, 2 and 3 in a suitable blender for 10-15 minutes. AddItem No. 4 and mix for 1-3 minutes. Fill the mixture into suitabletwo-piece hard gelatin capsules on a suitable encapsulating machine.

While the present invention has been described in conjunction with thespecific embodiments set forth above, many alternatives, modificationsand variations thereof will be apparent to those of ordinary skill inthe art. All such alternatives, modifications and variations am intendedto fall within the spirit and scope of the present invention.

What is claimed is:
 1. A compound of the formula: ##STR131## or apharmaceutically acceptable salt or solvate thereof, wherein: (1) R¹ isa group selected from: ##STR132## R² is selected from: (1) H,(2) C₁ toC₈ alkyl, (3) C₂ to C₈ alkenyl, (4) C₂ to C₈ alkynyl, (5) ##STR133##wherein said alkyl, alkenyl, or alkynyl is optionally substituted withone or more groups independently selected from: (a) aryl, aralkyl,heteroaryl, heteroarylalkyl or heterocycloalkyl; said aryl, aralkyl,heteroaryl, heteroarylalkyl or heterocycloalkyl optionally substitutedwith one or more groups independentlyselected from: (1) C₁ to C₄ alkyl,(2) (CH₂)_(t) OR⁸ wherein t is 1 to 4, (3) (CH₂)_(t) NR⁸ R⁹ wherein t is1 to 4, or (4) halogen,(b) C₃ to C₆ cycloalkyl, (c) --OR⁸, (d) --SR⁸,(e) --S(O)R⁸, (f) --SO² R⁸, (g) --NR⁸ R⁹, (h) ##STR134## R³ is selectedfrom H, halogen or C₁ to C₆ alkyl; R⁴ is selected from H, halogen or C₁to C₆ alkyl; R⁵ is selected from: H, ##STR135## R⁶ is selected from H orC₁ to C₆ alkyl; R⁷ is selected from H, C₁ to C₆ alkyl, haloalkyl, or--C(O)R¹¹ wherein R¹¹ is selected from C₁ to C₆ alkyl, C₁ to C₆ alkoxyor --NHR¹² (wherein R¹² is C₁ to C₆ alkyl or H), or R⁷ is an acylradical of a naturally occurring amino acid; R⁸, R⁹ and R¹⁰ areindependently selected from H, C₁ to C₄ alkyl, C₃ to C₆ cycloalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, aryl or aralkyl; saidalkyl, cycloalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, arylor aralkyl are optionally substituted with C₁ to C₄ alkoxy, aryl,heteroaryl, heterocycloalkyl, cyclopropyl, halogen, --OH, --C(O)R¹³,--SO₂ R¹³, or --NR¹⁴ R¹⁵ wherein R¹³ is selected from C₁ to C₄ alkyl oraralkyl, and wherein R¹⁴ and R¹⁵ are independently selected from H, C₁to C₄ alkyl or aralkyl; with the proviso that R⁸ is not H insubstituents (e), (f) or (k), and with the proviso that R⁹ is not H insubstituent (h) or (n), and with the proviso that R⁸, R⁹, or R¹⁰ is not--CH₂ OH or --CH₂ NR¹⁴ R¹⁵ when R⁸, R⁹, or R¹⁰ is directly attached to aheteroatom; optionally, when R⁸ and R⁹ are bound to the same nitrogen,R⁸ and R⁹, together with the nitrogen to which they are bound, form a 5to 7 membered heterocycloalkyl ring; optionally, when R⁹ and R¹⁰ arebound to the same nitrogen, R⁹ and R¹⁰, together with the nitrogen towhich they are bound, form a 5 to 7 membered heterocycloalkyl ring;--represents an optional bond; W is selected from CH when the optionalbond is present, or O, S or CH₂ when the optional bond is absent; X isN; and Y is selected from N or CH.
 2. The compound of claim 1 wherein R³and R⁴ are halogen.
 3. The compound of claim 2 wherein R³ is Cl and R⁴is Br.
 4. The compound of claim 1 wherein Y is N.
 5. The compound ofclaim 1 wherein R¹ is selected from ##STR136##
 6. The compound of claim5 wherein R⁵ is H.
 7. The compound of claim 1 wherein R² is selectedfrom H, --C₄ H₉, --CH₂ C₆ H₅, --CH₂ CH₂ OCH₃, --CH₂ CH₂ SCH₃, --CH₂ CH₂O-n-C₃ H₇, --CH₂ CH₂ CH₂ OCH₃, ##STR137##
 8. The compound of claim 1wherein W is CH or CH₂, and Y is N.
 9. The compound of claim 8 whereinR³ is Cl and R⁴ is Br.
 10. The compound of claim 9 wherein R¹ isselected from ##STR138## wherein R⁵ is H, and R² is selected from H,--C₄ H₉, --CH₂ C₆ H₅, --CH₂ CH₂ OCH₃, --CH₂ CH₂ SCH₃, --CH₂ CH₂ O-n-C₃H₇, --CH₂ CH₂ CH₂ OCH₃, ##STR139##
 11. The compound of claim 10 havingthe formula: ##STR140##
 12. A method for inhibiting the abnormal growthof cells comprising administering an effective amount of a compound ofclaim
 1. 13. The method of claim 12 wherein the the cells inhibited aretumor cells expressing an activated ras oncogene.
 14. The method ofclaim 12 wherein the cells inhibited are pancreatic tumor cells, lungcancer cells, myeloid leukemia tumor cells, thyroid follicular tumorcells, myelodysplastic tumor cells, epidermal carcinoma tumor cells,bladder carcinoma tumor cells or colon tumors cells.
 15. The method ofclaim 12 wherein the inhibition of the abnormal growth of cells occursby the inhibition of farnesyl protein transferase.
 16. The method ofclaim 12 wherein the inhibition is of tumor cells wherein the Rasprotein is activated as a result of oncogenic mutation in genes otherthan the Ras gene.
 17. A pharmaceutical composition for inhibiting theabnormal growth of cells comprising an effective amount of compound ofclaim 1 in combination with a pharmaceutically acceptable carrier. 18.The compound of claim 1 wherein R¹ is selected from: ##STR141##
 19. Thecompound of claim 1 wherein R¹ is selected from: ##STR142##
 20. Thecompound of claim 1 wherein R¹ is selected from: ##STR143##